The proposed research seeks to decipher the mechanism of viral RNA replication, a key step in production of progeny viruses and their spread to uninfected cells. A greater understanding of these mechanisms will facilitate our design of novel antiviral therapies. We study this process in the nodavirus Nodamura virus (NoV) so that we can harness its ability to replicate its positive-strand RNA genome to tremendously high levels in a wide range of host cells NoV provides an excellent model to study more complex, positive-strand RNA viruses pathogenic to humans. Although this model virus has not yet been associated firmly with human disease, recent studies have linked novel NoV-like viruses to human diarrheal or paralytic illnesses. Thus, we propose that NoV could serve as a model to understand the diseases caused by human Coxsackie viruses and newly designed experiments compare NoV replication complex assembly with that of the enterovirus Coxsackie A virus 21 (CVA21). Replication of NoV and CVA21 RNA occurs via the synthesis of complementary negative strand replication intermediates, which are used as templates for synthesis of additional positive strands. Both reactions are catalyzed by a virus-encoded RNA-dependent RNA polymerase (RdRp). Initiation of viral RNA replication in a host cell requires recognition of the RNA template by the RdRp and assembly of membrane- bound RNA replication complexes (RC's), the sites of RNA replication in the cell. The proposed studies focus on two major areas that impact RC assembly: 1) structural requirements of template recognition by the RdRp and subsequent template recruitment to the RC and 2) the role of RdRp post-translational modification in RC assembly. We hypothesize that recruitment of the required components is controlled by structure-dependent RNA-protein interactions between the RNA template and viral (and/or cellular) proteins and by post- translational modification of the RdRp. We will use a combined genetic and biochemical approach to make mutations that effect RdRp-binding to RNA templates or prevent modification of the RdRp and test their effects on RNA replication, RNA binding studies to measure interactions between the RNA template and the RdRp, microscopy studies to study changes to subcellular localization of the RdRp in response to mutagenesis, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the amino acids in the RdRp that are post-transnationally modified. We will also design a new RNA structure prediction tool to facilitate identification of long-range base pairing interactions within more complex viral RNA genomes. We propose the following Specific Aims: 1) Define the role of RNA structural elements in recognition by the RdRp and recruitment of RNA templates into mitochondrial RC's and 2) Determine the role of post-translational modification of the nodavirus RdRp in the formation of and recruitment of other proteins to membrane-bound RC's. The results of these studies will provide insight into viral RNA replication mechanisms and identify new targets for antiviral therapies.